Proceedings of the 20th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, Vol. 20, No 6, 1998
DETECTION AND CHARACTERIZATION OF
EVOKED QUANTAL DEPOLARIZATIONS IN SMOOTH MUSCLE
Rohit Manchanda* and K. Venkateswarlu, School of Biomedical Engineering,
Indian Institute of Technology-Bombay, Powai, Mumbai - 400 076, INDIA,
*E-mail: [email protected]
a larger evoked depolarization which triggers excitation [ll.
In smooth muscle, however, there has been no direct evidence
that the quantal depolarization, the spontaneous excitatory
The relationship between spontaneouslygenerated quantal
junction potentials (sEJP), is the fundamental unit of the
depolarizations and the nerve-stimulation elicited evoked
evoked EJP (eEJP) produced by nerve stimulation [l], [2]. A
depolarizations during neurotransmission in smooth muscle
major problem is that the sEJP is relatively brief in duration
organs has remained enigmatic. This issue was explored by
(50-150 ms) compared with the eEJP (600-10oO ms).
studying the effects of a presumptive intercellularuncoupling
Secondly, the amplitude of the eEJP is not an integral
agent, 1-heptanol, on the synaptic or "junction" potentials of
multiple of the s a p . Furthermore, sEJP-like depolarizations
smooth muscle, using intracellularrecording. In the guinea-pig have not been observed to occur following nerve stimulation.
vas deferens, heptanol was found to suppress the nerve- These incongruities are believed to arise from the fact that
stimulation evoked excitatory junction potential (eEJP) smooth muscle cells are electrically coupled to one another in
reversibly while the spontaneous EJPs (sEJPs) persisted.
a "three-dimensional syncytium" via intercellular gap junctions
However, during suppression of the eEJP in certain cells
(active cells), relatively brief stimulus-locked depolarizations [31,c41.
Evidently, it would be of interest to explore the effects on
still occurred intermittently, even though the prolonged
the junction potentials of uncoupling the smooth muscle cells
depolarization of the eEJP was abolished. Analysis of these
from one another electrically. The possibility for conducting
heptanol-resistant evoked depolarizations revealed a close such inquiries has arisen recently owing to the discovery of
similarity with the properties of sEJPs. Since the sEJP is chemical agents that appear specifically to uncouple smooth
thought to represent the quantal unit of neurofxansmitter action
muscle cells from one another functionally, leaving other
in smooth muscle, the heptanol-resistant depolarizationshave
properties unaffected, one such agent being the aliphatic
been termed quantal EJPs (qEJPs). Our results show for the
alcohol 1-hepranol [5].In recent work, we have studied the
first time that the unitary depolarization underlying the
gross effects of 1-heptanol on eEJPs of the smooth muscle of
syncytial eEJP of smooth muscle is an sEJP-like event. We
the guinea-pig vas deferens, using intracellular recording
discuss the significance of this fmding in terms of the
techniques [6]. We report here that in some cells, the effects
electrical behaviour of syncytialsmooth muscle, and speculate
of heptanol have allowed us to observe directly the quantal
on the possible biophysical effect by which heptanol action
depolarizations underlying the eEJP, which we term quantal
may give rise to qEJps.
EJPs (qEJPs), and to establish the quantal relationship
between the eEJP and the sEJP. We discuss the implications
KEYWORDS: Electrical syncytium, quantal depolarizations, of our findings in relation to the electrical input-output
relations of smooth muscle during neurotransmission and the
smooth muscle, electrical uncoupling., heptanol.
possible mode of action by which heptanol reveals the quantal
evoked depolarizations in this tissue.
I. INTRODUCTION
ABSTRACT
The electrical behaviour of smooth muscle during
neurotransmission from autonomic nerves is not clearly
understood. One of the most puzzling features of
neurotransmission in an electrically syncytial tissue such as
smooth muscle is the uncertain relationship between the
neurotransmitter-activated depolarizations that are generated
spontaneously and those that arise following nerve
stimulation. At other synapses, nerve cells release
neurotransmitter in quantal packets. Spontaneous release is
monoquantal and produces a quantal depolarization in the
target cell. Following nerve stimulation, neurotransmitter
release is multi-quantal and the summation of quanta produces
0-7803-5164-9/98/$10.000 1998 IEEE
II. METHODS
Vasa deferentia along with the innervating branch of the
hypogastric nerve were dissected out from exsanguinatedmale
Hartley guinea-pigs weighing 400-600 g. The vas was pinned
out on the silicone rubber base of a Perspex organ bath
(capacity 7-8 ml.). It was continuously superfused with Krebs
solution (composition in mM: NaC1, 118.4; KC1, 4.7; MgCl,,
1.2; CaCl,, 2.5; NaHCO,, 25.0; NaH,PO,, 0.4; Glucose, 11.1)
flowing under gravity at 2 - 3 ml/min. The solution was
bubbled with a gas mixture of 95% 0, and 5% CO, to
maintain its pH between 7.3 and 7.4. The surface connective
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A
S
B
Fig. 1. Emergence of rapid evoked depolarizations(qEJPs, asterisks) in active cells following suppression of eEJPs by 2.0mM heptanol.
Three records superimposed in each panel. A: control,B: after inhibition of e E P s to 10% of control amplitude. Filled circle: SEW,
S: stimulus artifact.
tissue was cut open carefully to facilitate intracellular
recordings from the outer longitudinal smooth muscle. A
Perspex chamber surrounding the organ bath was used to
achieve control of temperature of the inner chamber at 35 37 C, employing electronically controlled resistive heating.
The hypogastric nerve was stimulated via bipolar AgAgCl ring electrodes with rectangular voltage pulses of
amplitude 2-10 V, pulse width 50-500 ps, at 0.7 Hz.
Intracellular recordings of membrane potential were obtained
by using glass high impedance micrcelectrodes (tip resistances
20 - 60 M a ) fdled with 3.0 M KCl [6]. Signals were led to
an intracellular electrometer (IE201, Wamer Instrument Corp.,
USA), displayed on a digital storage oscilloscope, low pass
filtered (-3 dl3 cutoff 1 kHz), and stored on tape for
subsequent collection and analysis on computer using an A/D
card (PCL 209, Dynalog Microsystems, Mumbai, India) and
customised software (SCAN: S y ~ p t i cCurrent Analysis
Programme, kindly supplied by Dr J Dempster, Strathclyde
University, Glasgow).
O
III. RESULTS
Depending upon the type of eEJP recorded, smooth
muscle cells of the guinea-pig vas deferens could be classified
broadly into two types: (i) "Active" cells (10-25%) which
probably receive direct input from close-contact varicosities
(CCVs), and are the loci in the tissue where neurotrmmitter
action induces depolarization directly; (ii) "Passive" cells (7590%) which receive no direct CCV input, the eEJP recorded
in these cells beiig picked up passively, mainly by virtue of
intercellular spread from active cells [2]. In passive cells,
superfusion of the presumptive gap junction uncoupling agent
1-heptanol at 1.0-2.0 mM suppressed eEJP amplitude
gradually and fmlly abolished it within 2-3 minutes without
affecting resting membrane potential [6].This suppressionwas
fully reversible on washing out the drug.
In active cells, however, once the eEJPs had been
suppressed to less than about 20%of their control amplitudes
by heptanol, a different pattern of stimulation-evoked activity
emerged. This consisted of short-duration stimulus-locked
depolarizations that occurred intermittently, commencing
within the same band of latencies as the eEJPs. The
emergence of these depolarizations, which we term quantal
EJPs (qEJPs) for reasons mentioned below, is shown in
Fig. 1.
Examples of individual qE3Ps are provided in Fig. 2A,
from a cell whose eEJPs were suppressed by heptanol almost
completely. Six successive stimulation-evoked records are
shown after the action of heptanol (note the absence of the
prolonged e m ) . The occurrence of stimulus locked qEPs is
indicated by asterisks. Three salient properties of the qEJPs
are noteworthy. (i) The occurrence of qEJPs is intermittent,
i.e. every stimulus does not succeed in evoking a qEJP
(Fig. 2A). (ii) qEJPs identical to each other can occur in the
same cell (Fig. 2B). (iii) Selected qEJPs and sEJPs recorded
in the same cell were virtually identical in all salient
properties (Fig. 2C). This correspondence was corifiied
statistically by comparing the rise time, decay time constant
( T ~ ~ and
) , duration of several qEJPs and SEWSrecorded
from the same cell (Table 1).
Table 1
Temporal properties of sEJPs and qEJPs compared statistically
(Student's t-test). Data are mean + s.em. (no. of observations).
P>O.1 indicates no significant difference.
~
~
~
Rise time (ms)
rhar (ms)
Duration(ms)
sEJPs
11.2+1.1(19)
30.2+1.3(9)
85.1+3.8(16)
qE3Ps
13.2t 0.8(14)
28.0+2.9(10)
95.6 5.3( 12)
P
I
0.178
I
0.508
I
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0.111
I
B
12mv
I..;"
d
100 ms
Fig. 2. A: qEJPs (asterisks), in a cell different ffom Fig. 1, after suppression of eEJP showing intermittence. Filled circle is an sEJP.
B: Identical qEJPs superimposed.C: Matching of qEJP with an sEJP (dotted line). Calibration for B and C common. S : stimulus artifact.
From previously established induction, these three
characteristics indicate strongly that the qEJPs are quantal
depolarizations underlying the eEJPs [21, 171.
sEJPs recorded in the presem of heptanol showed no
alteration compared with control sEJPs. As shown in Fig. 3A,
there was no effect on time cowses of sEJPs. Their frequency
of occurrence also did not change, when monitored over
longer periods (3-4 min.). Finally, the amplitude histogam of
sEJPs was also left unaffected by heptanol (Fig. 3 B).
IV.DISCUSSION
The effects of heptanol on eEJPs provide insight into the
quantal relation between the eEJP and sEJP. The fundamental
quantal contributions to the eEJP have been suggested to be
identical to SEWS,based on different lines of argument [2].
However, their occurrence has never been directly observed,
because they are submerged in the general syncytial
depolarization of the eEJP. Our results show that these quantal
evoked depolarizations (qEJPs) can be detected following the
application of heptanol, a chemical reported to be an
intercellular electricaluncoupler [5]. Their observedproperties
(stimulus-locking; intermittence; repetition of a particular
event) correspond closely to those of evoked quantal release
determined by other methods [2], 171. Therefore, qEJPs reflect
evoked quantal release events occurring in the electrical
vicinity of the recording microelectrode. Since various
characteristics of these qEJPs, e.g. range of amplitudes and
time courses, are essentially the same as those of sEJPs (Fig.
2), this corroborates the suggestion that the basic evoked
quantal depolarization is an sEJP-like event. Therefore this
feature of neurotransmission in smooth muscle can be
concluded to be essentially similar to that at other synapses
[8], and the apparent difference seems to arise from the
syncytial properties of this tissue, which endows it with
unusual electrical properties [l].
A question of central interest is the mechanism by which
heptanol ''unmasks'' the qEJP from the eEJP. Heptanol is
suggested to specifically block gap junctional channels
mediating electrical continuity [5], thus uncoupling syncytial
cells from one another electrically. This effect may allow the
resolution of transmitter action at individual neuromuscular
junctions as follows. When an active cell has been uncoupled
from its neighbows, depolarizations generated remotely, that
are normally propagated passively to that cell, may no longer
be recorded in it. Therefore the background depolarization of
the eEJP m a y be removed, leaving behind only locally
generated quantal depolarizations caused by activation of
nearby transmitter release sites.
However if this explanation is to be accepted it must also
account consistently for the observations on SEES in the
presence of heptanol, which show no alteration compared with
control sEJPs. This is contrary to expectation. Theoretically,
sEJPs are predicted to be prolonged when cells are uncoupled
from one another [9], since the brief time come of the sEJP
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A
"n
B
n
a
b
60
60
Heptanol
Control
40
20
1
0
L3
5
7
9
1
1
1
O
1
Amplitude (mv)
Amplitude (mv)
Fig. 3. A: sEJPs (3-5 traces superimposed) control (a) and in the presence of 2.0mM heptanol (b). B: Amplitude histograms of sEJPs.
Note that the temporal properties and amplitude histograms of sEJPs are not affated by heptanol.
relative to the eEJP is thought to result directly from
extensive syncytial coupling [9].The amplitude histogram of
sEJPs would also be expected to be changed, reflecting fewer
low-amplitude and greater numbers of large-amplitude events
[lo].
Since experimental observationsdo not match predictions
about sEJPs based upon cell-to-cell uncoupling an explanation
for the suppression of eEJPs by heptanol, and the emergence
of qHPs, on this basis must at present remain speculative.
The exact mechanism of action of heptanol at these
neuromuscular junctions merits further exploration. One
possibility is that heptanol inhibits, specifically, the
stimulution-evoked release of neurotransmitter from the
autonomic innervation. The results are compatible with this
possibility. In this event, a lower net syncytial depolarization
would result, explaining the suppression of the e m . Since
postjunctional syncytial behaviour still prevails, sEJPs would
be left unaffected. Furthermore, at low prejunctional densities
of release, there is evidence that evoked depolarizations
should also be briefer [111, explaining the rapidity of qEJPs.
If conoborated, this mode of action of heptanol would
represent a novel, hitherto unsuspected biophysical mechanism
of interference, and would merit detailed scrutiny.
ACKNOWLEDGEMENTS
Financial support from the Department of Science &
Technology, India,under Project SP/SO/NO6/93, is gratefully
acknowledged.
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